Recent studies have shown that N-nitroso carcinogens, in particular N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), rapidly increase cGMP in intact cells and stimulate guanylate cyclase activity in tissue homogenates. Studies correlating electron spin resonance (ESR) measurements with enzymatic activity in a partially purified system have indicated that formation of the paramagnetic NO-ferrous heme complex is an obligate step in guanylate cyclase activation by MNNG and related agents. Stimulatory effects of NO-heme derivatives on guanylate cyclase appear to be expressed through redox or free radical reactions involving tissue sulfhydryl groups, with SH to S-S transformation a possible means of enzyme activation. The overall objectives of the proposed work are: 1) Examination of the mechanisms by which MNNG and related agents stimulate guanylate cyclase activity in a highly purified system. Emphasis will be placed on determining whether heme is a prosthetic group of gyanylate cyclase. 2) Examination by ESR spectroscopy of the structural and functional interaction of NO-heme with guanylate cyclase in the presence and absence of substrate and divalent cations. 3) Study of the effects of activated forms of acetylaminofluorene (AAF) on guanylate cyclase activity, as a potential model for the action of other procarcinogens. AAF is converted to N-hydroxylamine, N-nitroso, hydroxyl and other radical species. These species may activate guanylate cyclase through a heme-dependent pathway and be identifiable by ESR spectroscopy. 4) Determination of the extent to which other oxidants and radical generators chemically unrelated to MNNG such as fatty acid peroxides and superoxide dismutase stimulate guanylate cyclase through heme-dependent mechanisms. 5) Determination of the free sulfhydryl content of the purified enzyme and the role of sulfhydryl groups in the expression of basal and stimulated cyclase activity.